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1 Supplementary Figure 1. Normal AMPAR-mediated fepsp input-output curve in CA3-Psen cdko mice. Input-output curves, which are plotted initial slopes of the evoked fepsp as function of the amplitude of the fiber volley (FV), are similar between CA3-Psen cdko and control mice. All data represent mean ± s.e.m. The number of slices (left) and mice (right) used in each experiment is indicated in parenthesis. 1
2 Supplementary Figure 2. Normal current-voltage relationship of AMPAR-mediated synaptic responses in CA3-Psen cdko mice. a. Representative action potential-evoked EPSCs recorded from CA1 pyramidal neurons at different holding potentials. Synaptic responses were elicited by focal stimulation and neurons were voltage clamped from -80 mv to +80 mv in 20 mv steps. b. The currentvoltage relationship in control and CA3-Psen cdko mice is plotted. The dashed line represents linear I- V relationship. The number of neurons (left) and mice (right) used in each experiment is indicated in parenthesis. 2
3 Supplementary Figure 3. Reduced paired-pulse facilitation in CA3-Psen cdko mice using intracellular recordings. a. Representative EPSC traces (average of 5 responses) evoked by two consecutive stimuli with a 40 ms inter-stimulus interval from control and CA3-Psen cdko mice. Synaptic responses were measured from voltage-clamped hippocampal CA1 neurons. b. Averaged paired-pulse ratio is plotted as a function of the inter-stimulus interval. All data represent mean ± s.e.m. *p < 0.05; **p < The number of neurons (left) and mice (right) used in each experiment is indicated in parenthesis. 3
4 Supplementary Figure 4. Reduced synaptic facilitation in CA3-Psen cdko mice. a. Representative EPSC traces (average of 2 responses) evoked by 1 and 20 Hz stimulus trains from control and CA3-Psen cdko mice. Synaptic responses were measured from voltage-clamped hippocampal CA1 neurons. b. Synaptic facilitation elicited by stimulus trains is impaired in a frequency-dependent manner in CA3-Psen cdko mice. Values of the EPSC amplitude are normalized to the amplitude of the first fepsp of the stimulus train in individual slices. The external calcium concentration is 2.6 mm. All data represent mean ± s.e.m. *p < 0.05; **p < 0.01; ***p< The number of neurons (left) and mice (right) used in each experiment is indicated in parenthesis. 4
5 Supplementary Figure 5. Calcium dependency of the synaptic facilitation defect in CA3-Psen cdko mice. The synaptic facilitation impairment is most severe at external calcium concentration of 0.5 mm (a), moderate at 5.0 mm (b) and absent at 7.5 mm (c) in CA3-Psen cdko mice. Values of the fepsp slope are normalized to the slope of the first fepsp of the stimulus train in individual slices. All data represent mean ± s.e.m. *p < 0.05; **p < 0.01.; ***p< The number of slices (left) and mice (right) used in each experiment is indicated in parenthesis. 5
6 Supplementary Figure 6. Normal synaptic facilitation in CA3-Psen1 cko and Psen2 -/- mice. Synaptic responses were elicited by stimulus trains at indicated frequencies. The external calcium concentration is 2.6 mm. Values of the fepsp slope are normalized to the slope of the first fepsp of the stimulus train in individual slices. All data represent mean ± s.e.m. *p < 0.05; **p < The number of slices (left) and mice (right) used in each experiment is indicated in parenthesis. 6
7 Supplementary Figure 7. Normal paired-pulse facilitation CA3-Psen1 cko and Psen2 -/- mice. Paired-pulse ratio (2 nd fepsp/1 st fepsp) was measured at different inter-stimulus intervals using field recording. Averaged paired-pulse ratio is plotted as a function of the inter-stimulus interval. All data represent mean ± s.e.m. *p < 0.05; **p < The number of slices (left) and mice (right) used in each experiment is indicated in parenthesis. 7
8 Supplementary Figure 8. No significant changes in recovery kinetics after depleting vesicle pools in CA3-Psen cdko mice. Recovery was measured by delivering a single action potential (test pulse) at different intervals following a train of stimuli (100 Hz, 0.5 s). The ratio of the EPSP slope elicited by the test pulse to the first response of the train is plotted to measure the percentage of recovery. Note that post-tetanic facilitation was observed at interval > 1s in both groups. All data represent mean ± s.e.m. The number of slices (left) and mice (right) used in each experiment is indicated in parenthesis. 8
9 Supplementary Figure 9. Normal miniature EPSCs in CA3-Psen cdko mice. a. Representative recording traces of miniature EPSCs in CA3-Psen cdko and control mice. b. Statistical analysis indicates normal mepsc frequency and amplitude in CA3-Psen cdko mice. Miniature EPSC was recorded from CA1 pyramidal neurons under whole-cell voltage clamp mode in the presence of picrotoxin (100 μm) and tetrodotoxin (1 μm). All data represent mean ± s.e.m. The number of neurons (left) and mice (right) used in each experiment is indicated in parenthesis. 9
10 Supplementary Figure 10. Normal current-voltage relationship of voltage-gated calcium current in CA3-Psen cdko mice. a. Representative traces of voltage-gated calcium currents elicited by membrane depolarization (-80 mv to -50 mv, -35 mv, -20 mv, -5 mv, 10 mv, +70 mv, +85 mv) in CA3 neurons from control and CA3-Psen cdko mice. Voltage gated sodium channels were blocked by extracellular tetrodotoxin (1 µm) and intracellular QX-314 (5 mm). Potassium channels were blocked by intracellular TEA-Cl (20 mm) and Cs + (110 mm). b. Plots of current-voltage relationship of VGCCs from CA3 neurons in control and CA3-Psen cdko mice. All data represent mean ± s.e.m. The number of neurons (left) and mice (right) used in each experiment is indicated in parenthesis. 10
11 Supplementary Figure 11. Dantrolene treatment mimics and occludes the effect of presenilin inactivation on frequency facilitation. Dantrolene (10 μm) treatment impairs synaptic facilitation in control slices at stimulus frequencies of 10 and 20 Hz, whereas it has no effect on the reduced synaptic facilitation in CA3-Psen cdko mice. Hippocampal slices were pre-treated with dantrolene (10 μm) for 1 hr prior to recording. The fepsp slopes are normalized to the 1 st response of the stimulus trains in individual slices. All data represent mean ± s.e.m. *p < The number of slices (left) and mice (right) used in each experiment is indicated in parenthesis. 11
12 Supplementary Figure 12. Xestospongin C treatment has no effect on synaptic facilitation in both control and CA3-Psen cdko mice. Hippocampal slices were pre-treated with Xestospongin C (1 μm) for 1hr prior to recording. Synaptic facilitation was measured by 10 stimuli at varied frequencies (1, 5, 10 and 20 Hz) in the presence of Xestospongin C. The fepsp slopes are normalized to the first response of the stimulus trains in individual slices. All data represent mean ± s.e.m. The number of slices (left) and mice (right) used in each experiment is indicated in parenthesis. 12
13 Supplementary Figure 13. Inactivation of presenilin expression in postnatal hippocampal neuronal cultures impairs PPF at glutamatergic synapses. a. Western blot showing effective loss of the C- terminal fragment (CTF) of presenilin1 in Cre-infected cultures. Neuronal cultures were infected with lentiviruses expressing either a functional Cre recombinase GFP fusion protein (Psen cdko) or a mutant Cre recombinase GFP fusion protein (Control) at DIV 2, and protein samples were collected at DIV 14. b. Impaired PPF in Psen cdko hippocampal cultures. Averaged paired-pulse ratio is plotted as a function of the inter-stimulus interval. Synaptic responses were evoked using a concentric stimulus electrode, and measured from voltage-clamped pyramidal neurons in the presence of 50 μm APV and 100 μm picrotoxin to block NMDAR and GABA type A receptor (GABAAR)-mediated responses, respectively. c. Normal synapse density in cultured Psen cdko neurons. Representative fluorescence images (left) of neurons from control or Psen cdko cultures show the EGFP fluorescence (green), MAP2 (blue) and synaptophysin (red) immunoreactivity as well as the merged view. Scale bar: 20 μm. Quantitative analysis of synaptophysin-positive synapses (per 50,000 μm 2 ) showed no difference between the control and the Psen cdko cultures (right). All data represent mean ± s.e.m. *p < 0.05; **p < The number of neurons (left) and independent cultures (right) used in each experiment is indicated in parenthesis. 13
14 Supplementary Figure 14. Blockade of RyR mimics and occludes the effect of presenilin inactivation on depolarization-induced [Ca 2+ ] i responses in hippocampal neuronal cultures. a. Representative 14
15 calcium images shows high potassium (80 mm KCl)-induced Ca 2+ responses in control and Psen cdko neurons among untreated, ryanodine-treated and Xestospongin C-treated groups. Neurons were loaded with Fura-2, and 340/380 ratios were measured to calculate [Ca 2+ ] i. KCl was added at t = 0. b-d. The representative traces (top) and the graphs (bottom) of KCl-induced Ca 2+ responses in control and Psen cdko neurons. Mean amplitude of basal [Ca 2+ ] i and the peak amplitude of KCl-induced [Ca 2+ ] i increases (Δ[Ca 2+ ] i ) in the absence (b) or presence of ryanodine (c, 100 μm 1 hr prior and during) or Xestospongin C (d, 1 μm 1 hr prior and during) are shown. All data represent mean ± s.e.m. *p < 0.05; **p < The number of neurons (left) and independent cultures (right) used in each experiment is indicated in parenthesis. 15
16 Supplementary Figure 15. A Model depicting the role of presenilin in the regulation of neurotransmitter release. Upon stimulation, calcium concentration at the presynaptic terminal is drastically elevated due to calcium influx through VGCCs and calcium induced calcium release (CICR) from intracellular stores, which is mediated through both ryanodine receptors and IP 3 receptors. Loss of presenilin function in the presynaptic terminal specifically disrupts ryanodine receptor-mediated Ca 2+ release from the ER store, thus reducing CICR and resulting in reduced increases of calcium induced by action potentials in the presynaptic terminal. This reduction in calcium increases impairs the probability of neurotransmitter release, and the decreased glutamate release causes LTP impairment in presenilindeficient presynaptic terminals. 16
17 Supplementary Figure 16. Subcellular localization of endogenous presenilin1. Subcellular fractionation of mouse cortices followed by western shows the presence of the N-terminal fragment (NTF) of presenilin1 in different subcellular fractions. While presenilin1 is most abundant in the Golgi and ER fraction (P3) and the synaptosomal membrane fraction (LP1), it is also present in the presynaptic synaptic vesicle fraction (LP2), which lacks the postsynaptic marker, PSD95, and is abundant in the presynaptic marker, SVP38. S1, cortex homogenate; S2, cytosol and light membrane; P2, crude synaptosomal fraction; S3, cytosolic fraction; P3, light membrane (Golgi and ER); LS1, synaptosomal cytosol; LP1, synaptosomal membrane fraction; LS2, LS1 fraction minus LP2 fraction; LP2, synaptic vesicle-enriched fraction. 17
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